A self-assembling ph-sensitive amphiphilic peptide for gene therapy

Abstract

The present invention discloses a pH-sensitive amphiphilic peptide capable of self- assembly, comprising the sequences: (XY)n-(XZ)m-(XY)n-X (I) (XZ)m-(XY)n-(XZ)m-X (II), where X is a positively charged hydrophilic amino acid, Y is an aromatic or aliphatic hydrophobic amino acid, and Z is a pH-sensitive amino acid. The peptide has a symmetric arrangement of amino acids and a total length of less than 50 amino acids, with n and m being integers between 1 and 20. The peptide is designed to deliver nucleic acids to eukaryotic cells with low cytotoxicity, offering potential applications in gene therapy.

Description

DescriptionTitle of Invention: A Self-Assembling pH-Sensitive Amphiphilic Peptide for Gene TherapyTechnical Field

[0001] The present invention relates to a gene delivery system, specifically selfassembling pH-sensitive amphiphilic peptides designed for efficient and low- toxicity delivery of nucleic acids into eukaryotic cells.Background Art

[0002] Gene delivery offers much potential for the prevention or treatment of a variety of hereditary or infectious diseases. Several methods have been studied and developed to achieve this purpose; the most important ones include viral and non- viral vectors. Viral vectors have been administered in several clinical phase trials with high efficiency and potency. However, unwanted immune responses and potential insertional mutagenesis are problems brought about by these methods. Consequently, non-viral vectors, such as polymers, polycations, liposomes, cationic lipids, carbon nanotubes, and protein- and peptide-based nanoparticles, have been considered in view of their relative safety, higher nucleic acid packaging ability, and ease of production. However, the transfection efficiency of non-viral vectors is generally lower at therapeutic doses, and many of these vectors have issues related to biodegradability, which may result in their accumulation in the body.

[0003] Polymers and polycations, such as polyethyleneimine (PEI) and poly-L-lysine, have been utilized to condense DNA into small particles, However, these molecules have higher toxicity, thus limiting their clinical application. Cationic lipids are difficult to synthesize, and do not have any features facilitating targeted delivery. The polymeric carriers can cause immunological responses and require complicated synthetic reactions in order to form homogeneous particles. Considering the above-mentioned challenges, new carriers with high efficiency of gene delivery and acceptable biocompatibility are urgently required to be developed. Peptides represent a relatively new class of non-viral vectors, which have lower potential for unwanted immune responses, are highly design-flexible, and have a established methods for synthesis. Being composed of natural aminoacids provides good biocompatibility for peptides, and their small size makes them relatively easy to design using computers and produce in large quantities.

[0004] Since peptides do not form tertiary structures, there is more freedom to choose the sequence of amino acids without disrupting the structural integrity. Secondly, a peptide synthesis process can incorporate the desired cellular receptor sequences, which would otherwise avoid the need for distinct steps of synthesis. Moreover, the choice of non-coded amino-acids expands the sequence space of synthetic peptides. A number of peptides have been under research as gene delivery vehicles, of which 8-mer and 9-mer polyarginines were found to be potent gene vectors in a variety of studies. Among them, the R9 peptide, a CPP containing nine linear arginine residues, represents one of the most used and efficient cellpenetrating peptides. Although it exhibited lower efficiency compared with other materials in gene delivery, R9 peptide is still one of the most frequently used and efficient cell-penetrating peptides (Minwoo Kim et. Al 2013).Summary of Invention

[0005] This summary is intended to provide an overview of the subject matter of the exemplary embodiments of the present disclosure, and is not intended to identify essential elements or key elements of the subject matter, nor is it intended to be used to determine the scope of the claimed implementations. The proper scope of the exemplary embodiments of the present disclosure may be ascertained from the claims set forth below in view of the detailed description below and the drawings.

[0006] In one general aspect, the present disclosure describes an exemplary selfassembling pH-sensitive amphiphilic peptide which is characterized by a symmetric arrangement of amino acids, and in the parallel or anti-parallel configuration, the hydrophobic moieties of the peptide structure faced each other.

[0007] The above general aspect may include one or more of the following features. In some embodiments, the hydrophilic amino acids are located in the C and N terminals of the peptide sequence, and alternately distributed within the peptide’s amino acid sequence. Furthermore, the designed peptide according to the present disclosure, comprises pH-sensitive and hydrophobic amino acids which are alternately placed in the sequence of the designed peptides. The hydrophobic and pH-sensitive amino acids are placed along the peptide sequence that either thepH-sensitive moiety is placed at the center of the designed peptide sequence, or the hydrophobic moiety is centered within the designed peptide sequence.

[0008] The above-mentioned general aspect may include one or more of the following features. In some exemplary embodiments, due to this arrangement of amino acids and the type of amino acids used in the sequence of the designed peptide structure, it imparts additional propertiessuch as self-assembling and co-assembling of the peptide, sensitivity to changes in specific pH range, uniform positive charge throughout the peptide structure, cell-penetrating capability, and the ability to bind to DNA phosphate groups.

[0009] According to some embodiments, the self-assembling pH-sensitive amphiphilic peptide may include at least one of the positively charged hydrophilic amino acids such as arginine (R), lysine (K), and Ornithine (O), aromatic or aliphatic hydrophobic amino acids such as: phenylalanine (F), tyrosine (Y), and tryptophan (W), valine(V), leucine(L), isoleucine(l), and pH-sensitive amino acids such as histidine (H). SEQ ID NOs: 1 to 24, are all the different possible states for the designed peptide in the present disclosure, which apply the mentioned features in previous paragraphs.

[0010] In another aspect, the self-assembling pH-sensitive amphiphilic peptide in this disclosure can deliver a group of nucleic acids to eukaryotic cells with low cytotoxicity. In one exemplary embodiment, SEQ ID NO.1 and SEQ ID NO.4 are designed to demonstrate the ability thereof in gene therapy applications.Technical Problem

[0011] Current non-viral gene delivery systems, while relatively safer than viral vectors, face significant limitations in terms of transfection efficiency, targeted delivery, and biocompatibility. Specifically, polymers and polycations exhibit high cytotoxicity, lack of targeting capabilities, and are challenging to synthesize, and many non- viral vectors have issues with biodegradability, leading to potential accumulation in the body. There is an urgent need for new gene delivery carriers that combine high efficiency, targeted delivery, and acceptable biocompatibility.Solution to Problem

[0012] The present disclosure addresses these issues by developing a selfassembling pH-sensitive amphiphilic peptide that effectively combines hightransfection efficiency with low cytotoxicity and improved biocompatibility. This innovative approach ensures targeted delivery of nucleic acids to eukaryotic cells, overcoming the limitations of current non-viral vectors and providing a more effective and safer alternative for gene therapy applications.Advantageous Effects of Invention

[0013] The designed peptide shows a significant improvement in delivering nucleic acids to eukaryotic cells, enhancing gene delivery outcomes with minimal cytotoxicity. Its structure composed of natural amino acids, ensures better biocompatibility, reducing the risk of adverse reactions in clinical settings. Additionally, its pH-sensitive self- and co- assembly with DNA enables enhanced delivery properties, which improves its overall functionality.

[0014] The peptide’s responsiveness to pH changes offers controlled release and activation, optimizing its therapeutic effectiveness. It’s straightforward synthesis process allows for scalability and widespread use, while the flexibility in amino acid sequence design ensures customization for various therapeutic applications. Additionally, the peptide’s structure, enhances biocompatibility and minimizes the risk of, making it a safer alternative to traditional viral vectors and therefore, for clinical application.Brief Description of Drawings

[0015] The drawing figures depict one or more implementations in accord with the present teachings, by way of example only, not by way of limitation. In the figures, like reference numerals refer to the same or similar elements.

[0016] [FIG.1] illustrates the process of the forming a designed peptide-pDNA (peptide-plasmid DNA) nanocomplex, in accordance with exemplary embodiments of the present disclosure.

[0017] [FIG. 2] shows the peptide sequence of R9, RFH (SEQ ID NO. 1), and RHF (SEQ ID NO. 4), along with molecular weight and their charge at neutral pH, consistent with one or more exemplary embodiments of the present disclosure.

[0018] [FIG. 3A] shows agarose gel electrophoresis diagram according to tendency of the R9, to co-assemble with pDNA at various nitrogen-to-phosphate (N / P) ratios atat pH « 4 and 7, consistent with one or more exemplary embodiments of the present disclosure.

[0019] [FIG. 3B] shows an agarose gel electrophoresis diagram illustrating the coassembly of RHF (SEQ ID NO. 4) with pDNA at different N / P ratios at pH ~ 4 and 7, consistent with one or more exemplary embodiments of the present disclosure.

[0020] [FIG. 3C] shows agarose gel electrophoresis diagram for the co-assembly of RFH (SEQ ID NO: 1) with pDNA at various N / P ratios (5, 10, 15, 20, 25, 30, 35, and 40) at pH « 4 and 7, consistent with one or more exemplary embodiments of the present disclosure.

[0021] [FIG. 4A] shows the morphology of peptide-pDNA nanocomplexes formed at an N / P ratio of 15using field emission scanning electron microscopy (FESEM), consistent with one or more exemplary embodiments of the present disclosure.

[0022] [FIG. 4B] shows the morphology of peptide-pDNA nanocomplexes formed at an N / P ratio of 30, using field emission scanning electron microscopy (FESEM), consistent with one or more exemplary embodiments of the present disclosure;

[0023] [FIG. 4C] presents the morphology peptide-pDNA nanocomplexes formed at an N / P ratio of 45, using field emission scanning electron microscopy (FESEM), consistent with one or more exemplary embodiments of the present disclosure;

[0024] [FIG. 5A] shows Luciferase assay on T293 cell lines, for comparing the transfection efficiency of the PEI nanocomplex with designed peptide (R9, RHF (SEQ ID NO. 4), and RFH (SEQ ID NO. 1)) nanocomplexes in N / P ratios of 20 to 60, consistent with one or more exemplary embodiments of the present disclosure;

[0025] [FIG. 5A] shows a Luciferase assay on Hela cell lines, comparing the transfection efficiency of the PEI nanocomplex with those of the designed peptide (R9, RHF (SEQ ID NO. 4), and RFH (SEQ ID NO. 1)) at N / P ratios of 20 to 60, consistent with one or more exemplary embodiments of the present disclosure;

[0026] [FIG. 6A] shows an MTT viability assay on T293 cell lines, comparing the cytotoxicity of the PEI nanocomplex with those of the designed peptide nanocomplexs (R9, RHF (SEQ ID NO. 4), and RFH (SEQ ID NO. 1)) ar N / P ratios of 20 to 60, consistent with one or more exemplary embodiments of the present disclosure;

[0027] [FIG. 6B] shows the MTT viability assay on Hela cell lines, comparing the cytotoxicity of the PEI nanocomplex with designed peptide nanocomplexes (R9, RHF (SEQ ID NO. 4), and RFH (SEQ ID NO. 1)) at N / P ratios of 20 to 60, consistent with one or more exemplary embodiments of the present disclosure.Description of Embodiments

[0028] The following detailed description is presented to enable a person skilled in the art to make and use the methods and devices disclosed in exemplary embodiments of the present disclosure. For purposes of explanation, specific nomenclature is set forth to provide a thorough understanding of the present disclosure. However, it will be apparent to one skilled in the art that these specific details are not required to practice the disclosed exemplary embodiments. Descriptions of specific exemplary embodiments are provided only as representative examples. Various modifications to the exemplary implementations will be readily apparent to one skilled in the art, and the general principles defined herein may be applied to other implementations and applications without departing from the scope of the present disclosure. The present disclosure is not intended to be limited to the implementations shown but is to be accorded the widest possible scope consistent with the principles and features disclosed herein.

[0029] May in one exemplary embodiment, the present invention discloses a selfassembling pH-sensitive amphiphilic peptide characterized by a mirroring symmetric arrangement of amino acids, with a length of less than 50 amino acids. The peptide, as disclosed herein, may have the capability to self-assemble and comprises sequences with the general formula as follow:(XY)n-(XZ)m-(XY)n-X (I)(XZ)m-(XY)n-(XZ)m-X (II)

[0030] In this formula, X may represent a positively charged hydrophilic amino acid, Y may represent an aromatic or aliphatic hydrophobic amino acid, Z may represent a pH-sensitive amino acid. The amphiphilic peptide is designed with these amino acids to achieve a balance of hydrophilic and hydrophobic properties, thereby facilitating its ability to self-assemble under different pH conditions. The peptide’s length and amino acid composition are selected to allow for its function in binding and delivering nucleic acids.

[0031] The amino acids used in the peptide sequence may be in D, L, or p forms. In some exemplary embodiment, the X amino acid may comprise at least one of arginine (R), lysine (K) or ornithine (O). In some embodiment, the Y amino acid may comprise at least one of phenylalanine (F), tyrosine (Y), tryptophan (W), valine(V), leucine(L), isoleucine(l), and alanine (A), while the Z in a preferred embodiment may comprise pH-sensitive amino acids, with histidine (H). Furthermore, the variables “n” and “m” represent repeating units and may be selected from integers between 1 and 20, wherein in certain embodiment, n is 1 and m is 2, or n is 2 and m is 1 . Specifically, X may be arginine (R) or lysine (K), Y may be phenylalanine (F), tryptophan (W) or tyrosine (Y), and Z may be the L form of histidine (H). In a preferred embodiment, the length of the designed peptide is about 6-10 amino acids.

[0032] The present invention may also disclose specific sequences (SEQ ID NOs 1- 24) for the self-assembling pH-sensitive amphiphilic peptide. The possible configurations of the arrangement of amino acid sequences (SEQ ID NOs 1-24) in the designed peptide strand in the present invention may include, but are not limited to, the following;RFRHRHRFR (SEQ ID NO: 1), RFRFRHRFRFR (SEQ ID NO: 2), RHRHRFRHRHR (SEQ ID NO: 3), RHRFRFRHR (SEQ ID NO: 4), RYRHRHRYR (SEQ ID NO: 5), RYRYRHRYRYR (SEQ ID NO: 6), RWRWRHRWRWR (SEQ ID NO: 7), RWRHRHRWR (SEQ ID NO: 8), RHRYRYRHR (SEQ ID NO: 9), RHRWRWRHR (SEQ ID NO: 10), RHRHRYRHRHR (SEQ ID NO: 11), RHRHRWRHRHR (SEQ ID NO: 12), KFKFKHKFKFK (SEQ ID NO: 13), KFKHKHKFK (SEQ ID NO: 14), KHKHKFKHKHK (SEQ ID NO: 15), KHKFKFKHK (SEQ ID NO: 16), KYKYKHKYKYK (SEQ ID NO: 17),KYKHKHKYK (SEQ ID NO: 18),KHKHKYKHKHK (SEQ ID NO: 19), KHKYKYLHK (SEQ ID NO: 20), KWKWKHKWKWK (SEQ ID NO: 21), KWKHKHKWK (SEQ ID NO: 22), KHKHKWKHKHK (SEQ ID NO: 23), KHKWKWKHK (SEQ ID NO: 24).

[0033] In one exemplary embodiment, the self-assembling pH-sensitive amphiphilic peptide may feature a uniform distribution of positive charges, attributable to the evenly and alternately spaced hydrophilic amino acids. These hydrophilic amino acids may be placed at the C and N terminal regions of the peptide. The peptide structure of the designed peptide may also include histidine amino acids, which may become charged at pH<6. In addition to hydrophobic amino acids that may localize in the central region of the peptide sequence. This configuration may result in a structure that is optimized for intermolecular interactions with negatively charged macromolecules like DNA, where hydrophobic interactions are crucial for peptide self-assembly.

[0034] In one exemplary embodiment, the mirroring symmetric arrangement of amino acids of the designed peptide is due to the alternate placement of the hydrophilic, hydrophobic, and pH-sensitive amino acids, where either the hydrophobic moiety or the pH-sensitive moiety may be placed in the center of the peptide sequence, and also positively charged moiety may be at the amine and carboxyl terminals along the peptide sequence.

[0035] The hydrophobic amino acids may be strategically positioned within the peptide sequence such that when two peptide molecules align either parallel or anti-parallel to each other, the hydrophobic moieties of the molecules may face each other in both orientations. This arrangement may be attributed to the symmetry and the specific distribution of hydrophobic and hydrophilic amino acids in the peptide sequence, facilitating intermolecular interactions.

[0036] Furthermore, the designed peptide, according to the present disclosure may be capable of binding to the phosphate groups of DNAs, through the charged amino acids located at both terminals. The charged amino acids, particularly those at theamine and carboxyl terminals, may interact with the negatively charged phosphate groups, allowing for nucleic acid binding and delivery.

[0037] In one exemplary embodiment, the designed peptide, according to the present disclosure may exhibits self-assembling properties due to the presence of hydrophobic amino acids with aromatic rings, which may promote TT-TT interactions. These interactions between aromatic rings can enhance the stability of the selfassembled peptide structure. In some exemplary implementations, the selfassembling and co-assembling properties of the designed peptide may be pH- sensitive due to the presence of amino acids, such as histidine, which possess a positive charge at pH<6 and repel each other, while at pH>6 they participate in TT- TT interactions.

[0038] Furthermore, the presence of positively charged amino acids positioned adjacent to aromatic amino acids within the peptide structure may impart cellpenetrating properties to the peptide. This arrangement may facilitate cellular uptake, further enhancing the peptide's potential for applications in delivering nucleic acids to eukaryotic cells.

[0039] In one embodiment, this disclosure may present two amphiphilic peptides, designed with the capability of self-assembly. The design method for those peptides may be based on a rational and bottom-up method. The two amphiphilic peptides may be introduced as representative designed samples, abbreviated as ‘RFH’ and ‘RHF’ (SEQ ID NO. 1 , and SEQ ID NO. 4, respectively). Their amino acid sequences and molecular weights are depicted in FIG. 2. These peptides may exhibit self-assembling properties, making them suitable for various applications, including potential use in nucleic acid delivery.Example 1 : Formation of Peptide-pDNA Nanocomplexes

[0040] In this example, the formation of peptide-plasmid DNA nanocomplex is described with the steps illustrated in FIG. 1. It should be noted that in this disclosure, whenever pDNA is mentioned, it means plasmid DNA. According to step 110 of FIG.1 , first, the R9, RFH (SEQ ID NO. 1), and RHF (SEQ ID NO. 4) peptides are designed. The synthesized peptide samples were received as lyophilized powders.

[0041] Next, in accordance with on FIG. 1 , step 120, the lyophilized powder was dissolved in a 100 microliter of a pharmaceutical water for injection. Then, the dissolved peptide samples were heated at about 70 °C diluted to a final volume of 1000 microliters. The pH of the solution was adjusted to 7, or 4, depending on the experimental conditions.

[0042] Subsequently, as shown in step 130, a commercially available plasmid containing the luciferase reporter gene containing the luciferase reporter gene, was used in the physicochemical and biological experiments. This plasmid was modified by inserting a cytomegalovirus (CMV) promoter. The plasmid DNA or pDNA was transformed into Escherichia coli TOP10 cells. The transformed cells were proliferated under the selective marker of an antibiotic, ampicillin. Then the transformed cells were purified by a commercially available plasmid extraction kit.

[0043] In step 140 of the FIG.1 , to form the nanocomplexes, 15 microliters of the peptide solutions with varying molar ratios of peptide nitrogen to pDNA phosphate (N / P) were mixed to 10 microliters of the plasmid DNA. The concentration of plasmid DNA is 0.1 pg / pL dissolved in TE buffer at pH = 8.5. The mixture was slowly pipetted for 20-25 seconds to thoroughly mix.

[0044] Finally, as illustrated in FIG.1 , step 150, the mixture was incubated at 4 °C for 25 minutes to form a peptide-pDNA nanocomplex. For comparison, PEI-pDNA nanocomplexes were also prepared with an N / P ratio of 10 using the same method. PEI used in these experiments had a branched structure and a molecular weight of 25 kDa.

[0045] FIG. 2, shows the molecular weight, charge in neutral pH, amino acid sequences, and structural formula of the control sample, R9 along with the designed peptide (SEQ ID NO. 1 and SEQ ID NO. 4) samples.Example 2: Agarose Gel Electrophoresis Assay on the PEI-pDNA, R9-pDNA, RFH (SEQ ID NO. 1)-pDNA, and RHF (SEQ ID NO. 4)-pDNA Nanocomplexes

[0046] In this example, agarose gel electrophoresis was used to evaluate the ability of the formed nanocomplexes to prevent pDNA electrophorus movement. Initially, the formed peptide-pDNA nanocomplexes were electrophoresed in the agarose gel 1 w / v% containing red gel, in a Tris-Borate (TBE) buffer with a current of 90 V for approximately 40 minutes. The agarose gel electrophoresis method evaluates boththe ability of peptides to protect DNA from external environment of the cells, and the strength of the peptide-pDNA binding within the formed nanocomplexes.

[0047] For evaluating process, pure pDNA was used as control sample. FIG. 3A, shows the tendency of the R9 peptide to bind to pDNA in different N / P ratios in the pH conditions of 4 and 7. FIG. 3B, shows the tendency of the RHF (SEQ ID NO. 4) peptide to bind to pDNA in different N / P ratios in the pH conditions of 4 and 7. FIG. 3C shows the tendency of the RFH (SEQ ID NO. 1) peptide to aggregate to pDNA in different N / P ratios (5, 10, 15, 20, 25, 30, 35, and 40) at both pH is about 4 and pH is about 7.

[0048] According to the results, the tendency of designed peptides (SEQ ID NO. 1 and SEQ ID NO.4) to co-assemble with pDNA in acidic and neutral condition was compared with R9 peptide in the agarose gel electrophoresis experiment. In this experiment, both peptides (SEQ ID NO. 1 and SEQ ID NO.4) were able to inhibit the electrophoretic movement of pDNA in neutral conditions with an N / P of 10 or less, similar to R9; However, under acidic conditions (PH « 4), RFH (SEQ ID NO. 1) lost its ability to bind to pDNA, which indicates a reduced capacity of the peptide to form a complex with pDNA in response to lower PH.

[0049] According to the results, the designed peptides (SEQ ID NO. 1 and SEQ ID NO. 4) were compared to the R9 peptide in terms of their ability to form nanocomplexes with pDNA under acidic and neutral conditions, and protect DNA against the external environment of cells. Both RFH (SEQ ID NO. 1) and RHF (SEQ ID NO. 4) inhibited the electrophoretic movement of pDNA in neutral conditions at an N / P ratio of 10 or lower, similar to R9. However, under acidic conditions (pH « 4), RFH (SEQ ID NO. 1) lost its ability to bind to pDNA, indicating a reduced capacity to form a complex in response to lower PH. While, RHF (SEQ ID NO. 4), retained this ability similar to R9 at pH « 4.Example 3: FESEM Imaging on the Peptide-pDNA Binding

[0050] In this example, the field emission scanning electron microscopy (FESEM) was used to evaluate the morphology of peptide-pDNA binding. To prepare the samples for imaging, 5 pL of the diluted nanocomplex mixture at three different N / P ratios (the molar ratio of the peptide nitrogen to the pDNA phosphate), equal to 15, 30,and 45, was applied onto a layer of fresh mica slide. The slides were placed near a flame for 30 minutes to allow the samples to dry completely.

[0051] After drying, to prevent dust particles from depositing on the slide, it was kept in a closed box. The prepared slides were coated with a carbon film, and their micrographs were observed using a field emission scanning electron microscope (FESEM), and finally, images with a magnification of 1.3 nm at 1 kV were obtained. The images captured the morphology of the peptide-pDNA nanocomplexes at different N / P ratios.

[0052] FIG. 4A, illustrates the field emission scanning electron microscopy (FESEM) images showing the morphology of peptide-pDNA nanocomplexes at an N / P ratio of 15. The images correspond to the peptides: 410: R9, 420: RHF (SEQ ID NO. 4), and 430: RFH (SEQ ID NO. 1). The R9 peptide predominantly forms spherical particles with diameters of less than 100 nm. In contrast, the RHF (SEQ ID NO. 4) peptide does not exhibit a distinct morphology at this N / P ratio, while the RFH (SEQ ID NO. 1) peptide forms spindle-shaped particles of varying sizes.

[0053] FIG. 4B, displays the FESEM images of the same peptides (410: R9, 420: RHF (SEQ ID NO. 4), and 430: RFH (SEQ ID NO. 1)) at an N / P ratio of 30. At this ratio, the R9 peptide continues to form spherical particles, and the density of these particles increases. The RHF (SEQ ID NO. 4) peptide begins to take on a spindlelike or "X"-shaped morphology, with dimensions of approximately 600 nm in length and 200 nm in width. The RFH (SEQ ID NO. 1) peptide forms smaller, well-defined spindle-shaped particles compared to its appearance at an N / P ratio of 15.

[0054] FIG. 4C, presents the FESEM images of the peptides, 410: R9, 420: RHF (SEQ ID NO. 4), and 430: RFH (SEQ ID NO. 1), at an N / P ratio of 45. The R9 peptide still forms spherical particles, with a further increase in density. RHF (SEQ ID NO. 4) shows predominantly "X"-shaped particles, which are more defined at this higher ratio, and appear to be formed from the accumulation of needle-like structures measuring over 1 pm in length and less than 100 nm in width. The RFH (SEQ ID NO. 1) peptide continues to form spindle-like particles, but their size has decreased to less than 1 pm, and needle-shaped particles become more visible as the N / P ratio increases.

[0055] According to the results shown in FIG. 4A, FIG. 4B, and FIG. 4C, the R9 peptide consistently forms spherical particles with a size of less than 100 nm at all N / P ratios. The density of spherical particles increases with the N / P ratio, indicating a greater aggregation of nanocomplexes at higher peptide concentrations.

[0056] In contrast, the particles formed by RHF (SEQ ID NO. 4) at an N / P ratio of 15 do not exhibit a specific or well-defined morphology. At an N / P ratio of 30, the RHF (SEQ ID NO. 4) particles begin to form spindle or "X"-shaped structures, with characteristic dimensions of approximately 600 nm in length and 200 nm in width. This spindle-like morphology becomes the predominant structure at an N / P ratio of 45, where the particles appear to consist of aggregated, needle-like formations over 1 pm in length and less than 100 nm in width.

[0057] Conversely, in the case of RFH (SEQ ID NO. 1), spindle-shaped particles dominate the morphology at all N / P ratios. At lower N / P ratios (15), these spindle- shaped particles are larger, with dimensions measuring several micrometers. As the N / P ratio increases, the size of the spindle-shaped particles decreases to less than 1 pm. Additionally, needle-shaped particles become more visible at higher N / P ratios (45), suggesting a transformation or fragmentation of the spindle structures as the peptide concentration increases.Example 4: Transfection Assay on T293 and Hela Cell Lines

[0058] In this example, a transfection assay has been used to evaluate the potency of the designed peptides in transferring nucleic acid to eukaryotic cells. In one exemplary embodiment of the present disclosure, R9 and poly-ethylenimine (PEI) were introduced as control samples. PEI is a non-degradable, non-viral vector that can accumulate in the body. It is highly efficient at condensing DNA into small particles, but this feature often increases its cytotoxicity, which can limit its clinical application. The R9 peptide composed of 9 linear arginine amino acids, making it one of the most common penetrating peptides.

[0059] Based on this assay, two cell lines comprising T293 and Hela were cultured in DMEM culture medium containing 10% fetal bovine serum (FBS) and 1 % penicillin / streptomycin. The cells were incubated in a cell culture incubator at 37 °C and 5% CO2. The cells were used before the fifth passage for the transfection assay. One day before the transfection assay, 96-well plates were treated withpoly-L-lysine to improve cell adhesion, subsequently, 15000-18000 cells were seeded into each well of the plates and incubated with 100 pL of DMEM containing 10% FBS for about 18 hours.

[0060] One the following day, the culture medium of the cells was removed, and the cells were washed once with PBS. Then, 75 pL of serum-free and antibiotic-free medium was added and cells were incubated for 2 hours to adapt to the new conditions. Next, 25 pL of the nanocomplex mixture was added to each well and incubated for 4-5 hours at 37 °C. After incubation, 100 pL of DMEM with 20% FBS was added until the medium contained 10% FBS in each well, and cells were incubated for 48 hours.

[0061] After the incubation period, the medium on the cells in each well was removed and the cells were washed with PBS. the expression of the reporter gene was measured using the luciferase assay kit, according to the manufacturer’s instructions. Light emission was measured by a cell imaging multi-mode reader and the results were reported in terms of PLLIs. The obtained results from the transfection results were divided by the total cell protein in each well and normalized in milligrams. Total cell protein was measured by the bicinchoninic acid (BCA) protein assay kit. PEI-pDNA nanocomplexes were used as a positive control for transfection, and pDNA was used as a control for DNA molecule transfection. Analyzing and charting the biological properties data was done by using data analyzing software. All of the quantitative data are the mean ± standard deviation of three independent replicates. ANOVA, a two-way analysis of variance followed by multiple comparisons, was performed to determine the effect of peptide type and N / P ratio on transfection and cytotoxicity in Hela and T293 cell lines.

[0062] FIG. 5A shows the results of the luciferase assay for the T293 cell lines, comparing the transfection efficiency of PEI-pDNA nanocomplex with the R9- pDNA, RHF (SEQ ID NO. 4)-pDNA, and RFH (SEQ ID NO. 1)-pDNA nanocomplexes at N / P ratios of 20, 30, 40, 50, and 60. Based on that, the expression level of the luciferase gene in the T293 cells that were transfected with PEI was higher than the expression level of the luciferase gene in the T293 cells that were transfected with the three peptides (R9, RFH (SEQ ID NO. 1), and RHF (SEQ ID NO. 4)) nanocomplexes, among the peptides, the designed peptide nanocomplexes (RHF (SEQ ID NO. 4)-pDNA and RFH (SEQ ID NO. 1)-pDNA)showed higher efficiency than the R9-pDNA nanocomplex. Moreover, the RFH (SEQ ID NO. 1) peptide nanocomplex (RFH (SEQ ID NO. 1)-pDNA) generally showed higher efficiency than the RHF (SEQ ID NO. 4) peptide nanocomplex (RHF (SEQ ID NO. 4)-pDNA) in gene delivery.

[0063] Similarly, FIG. 5B shows the results of the luciferase assay on the Hela cell lines, comparing the transfection efficiency of PEI-pDNA nanocomplexes with the R9-pDNA, RHF (SEQ ID NO. 4)-pDNA, and RFH (SEQ ID NO. 1)-pDNA nanocomplexes in the N / P ratios of 20, 30, 40, 50, and 60. Based on that, the expression level of the luciferase gene in the Hela cell lines, that were transfected with PEI was again higher than that observed with the peptide nanocomplexes. However, the designed peptide nanocomplexes (RHF (SEQ ID NO. 4)-pDNA and RFH (SEQ ID NO. 1)-pDNA) showed higher efficiency than the R9-pDNA nanocomplexes, similarly, the RFH (SEQ ID NO. 1) peptide nanocomplex (RFH (SEQ ID NO. 1)-pDNA) generally showed higher efficiency than the RHF (SEQ ID NO. 4) peptide nanocomplex (RHF (SEQ ID NO. 4)-pDNA) in transfection.

[0064] In comparison of FIG. 5A and FIG. 5B, it is evident that the luciferase expression level in the T293 cells was higher than the Hela cell lines for all the N / P ratios. However, the difference in efficiency between the designed peptides and PEI, were calculated based on the logarithm of the amount of emitted light divided by milligrams of cellular protein, was not statistically significant in the N / P ratio equal or more than 40.Example 5: MTT Cytotoxicity Assay on T293 and Hela Cell lines

[0065] In this example, the cytotoxicity of the peptide-pDNA and PEI-pDNA nanocomplexes was measured on the T293 and Hela cell lines by MTT method. The cell lines were treated with the sample and the control nanocomplexes similar to the transfection assay. After 48 hours, the medium was replaced with 100 pL of fresh medium. Then, 10 pL of MTT solution, which comprised 5 mg of the MTT powder in 1 mL of PBS buffer, was added to each well and incubated for 4 hours. After that, 75 pL of the earlier solution was removed and 75 pL DMSO was added. Finally, the amount of absorption was measured by an Epoch microplate reader at a wavelength of about 570 nm.

[0066] FIG. 6A shows the results of the MTT cell viability assay on T293 cells, investigating the cytotoxicity of the PEI-pDNA nanocomplex in comparison to R9- pDNA, RHF (SEQ ID NO. 4)-pDNA, and RFH (SEQ ID NO. 1)-pDNA at N / P ratios of 20, 30, 40, 50, and 60. Naive cells (untreated) were used as the negative control for the viability assay. The results demonstrate that the R9, RHF (SEQ ID NO. 4), and RFH (SEQ ID NO. 1) peptides exhibit lower cytotoxicity than PEI at all N / P ratios, indicating their reduced impact on cell viability in T293 cells.

[0067] Similarly, FIG. 6B shows the results of the MTT cell viability assay on Hela cells, focusing on the cytotoxicity of the PEI-pDNA nanocomplex in comparison to R9-pDNA, RHF (SEQ ID NO. 4)-pDNA, and RFH (SEQ ID NO. 1)-pDNA at the same N / P ratios. Naive cells (untreated) were used as the negative control for the viability assay. The data indicate that the R9, RHF (SEQ ID NO. 4), and RFH (SEQ ID NO. 1) peptides exhibit lower cytotoxicity than PEI at all N / P ratios, confirming their reduced impact on cell viability in Hela cells.Industrial Applicability

[0068] The disclosed pH-sensitive amphiphilic peptide demonstrates significant industrial applicability in the fields of gene therapy and drug delivery. Its selfassembling properties, biocompatibility, and minimal cytotoxicity make it an ideal candidate for delivering nucleic acids to eukaryotic cells in clinical settings. The peptide’s ability to respond to pH changes allows for controlled release and targeted activation, enhancing therapeutic precision and effectiveness. Furthermore, the peptide's customizable amino acid sequence enables adaptation for various therapeutic applications, such as cancer treatment, genetic disorders, and other diseases requiring targeted gene delivery, positioning it as a safer and more efficient alternative to viral vectors.Sequence Listing Free Text

[0069] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on 2024.09.14, is named -_SequenceListing.xml and is - - kilobytes in size.

Claims

Claims

1. A pH-sensitive amphiphilic peptide with capability to self-assemble, comprising following sequences:(XY)n-(XZ)m-(XY)n- X (I)(XZ)m-(XY)n-(XZ)m-X (II)Wherein X is a positively charged hydrophilic amino acid, Y is an aromatic or aliphatic hydrophobic amino acid, and Z is a pH-sensitive amino acid, wherein the pH-sensitive amphiphilic peptide has a mirroring symmetric arrangement of amino acids, having a length of less than 50 amino acids.

2. The pH-sensitive amphiphilic peptide according to claim 1 , wherein n and m are repeating units selected from integers between 1 and 20.

3. The pH-sensitive amphiphilic peptide according to claim 1 and 2, wherein the n is 1 and the m is 2, or the n is 2 and the m is 1.

4. The pH-sensitive amphiphilic peptide according to claim 1 , wherein the X, Y, and Z amino acids comprising amino acids having D, L, or p forms.

5. The pH-sensitive amphiphilic peptide according to claim 1 and 4, wherein the X comprising at least one of arginine, lysine, and ornithine amino acids.

6. The pH-sensitive amphiphilic peptide according to claim 5, wherein the X is arginine amino acid.

7. The pH-sensitive amphiphilic peptide according to claim 1 and 4, wherein the Y comprising at least one of phenylalanine, tyrosine, tryptophane, valine, leucine, isoleucine, and alanine amino acids.

8. The pH-sensitive amphiphilic peptide according to claim 7, wherein the Y is phenylalanine amino acid.

9. The pH-sensitive amphiphilic peptide according to claim 1 and 4, wherein the Z is histidine amino acid.

10. The pH-sensitive amphiphilic peptide according to claim 1 , wherein the pH-sensitive amphiphilic peptide delivers any kind of nucleic acids to eukaryotic cells.]

Images